SEA-PHAGES and SEA-GENES: Advancing Virology and Science Education.

Research opportunities for undergraduate students are strongly advantageous, but implementation at a large scale presents numerous challenges. The enormous diversity of the bacteriophage population and a supportive programmatic structure provide opportunities to engage early-career undergraduates in phage discovery, genomics, and genetics. The Science Education Alliance (SEA) is an inclusive Research-Education Community (iREC) providing centralized programmatic support for students and faculty without prior experience in virology at institutions from community colleges to research-active universities to participate in two course-based projects, SEA-PHAGES (SEA Phage Hunters Advancing Genomic and Evolutionary Science) and SEA-GENES (SEA Gene-function Exploration by a Network of Emerging Scientists). Since 2008, the SEA has supported more than 50,000 undergraduate researchers who have isolated more than 23,000 bacteriophages of which more than 4,500 are fully sequenced and annotated. Students have functionally characterized hundreds of phage genes, and the phage collection has fueled the therapeutic use of phages for treatment of Mycobacterium infections. Participation in the SEA promotes student persistence in science education, and its inclusivity promotes a more equitable scientific community.

Bacteriophage tRNA-dependent lysogeny: requirement of phage-encoded tRNA genes for establishment of lysogeny.

Bacteriophages are large and diverse components of the biosphere, and many phages are temperate. Upon infection, temperate phages can establish lysogeny in which a prophage is typically integrated into the bacterial chromosome. Here, we describe the phenomenon of tRNA-dependent lysogeny, a previously unrecognized behavior of some temperate phages. tRNA-dependent lysogeny is characterized by two unusual features. First, a phage-encoded tyrosine family integrase mediates site-specific recombination between a phage attP site and a bacterial attB site overlapping a host tRNA gene. However, attP and attB share only a short (~10 bp) common core such that a functional tRNA is not reconstructed upon integration. Second, the phage encodes a tRNA of the same isotype as the disrupted but essential host tRNA, complementing its loss, and consequently is required for the survival of lysogenic progeny. As expected, an integrase-defective phage mutant forms turbid plaques, and bacterial progeny are immune to superinfection, but they lack stability, and the prophage is rapidly lost. In contrast, a tRNA-defective phage mutant forms clear plaques and more closely resembles a repressor mutant, and lysogens are recovered only at very low frequency through the use of secondary attachment sites elsewhere in the host genome. Integration-proficient plasmids derived from these phages must also carry a cognate phage tRNA gene for efficient integration, and these may be useful tools for mycobacterial genetics. We show that tRNA-dependent lysogeny is used by phages within multiple different groups of related viruses and may be prevalent elsewhere in the broader phage community.IMPORTANCEBacteriophages are the most numerous biological entities in the biosphere, and a substantial proportion of phages are temperate, forming stable lysogens in which a prophage copy of the genome integrates into the bacterial chromosome. Many phages encode a variety of tRNA genes whose roles are poorly understood, although it has been proposed that they enhance translational efficiencies in lytic growth or that they counteract host defenses that degrade host tRNAs. Here, we show that phage-encoded tRNAs play key roles in the establishment of lysogeny of some temperate phages. They do so by compensating for the loss of tRNA function when phages integrate at an attB site overlapping a tRNA gene but fail to reconstruct the tRNA at the attachment junction. In this system of tRNA-dependent lysogeny, the phage-encoded tRNA is required for lysogeny, and deletion of the phage tRNA gives rise to a clear plaque phenotype and obligate lytic growth.

Bacteriophage infection and killing of intracellular Mycobacterium abscessus.

IMPORTANCE: As we rapidly approach a post-antibiotic era, bacteriophage (phage) therapy may offer a solution for treating drug-resistant bacteria. Mycobacterium abscessus is an emerging, multidrug-resistant pathogen that causes disease in people with cystic fibrosis, chronic obstructive pulmonary disease, and other underlying lung diseases. M. abscessus can survive inside host cells, a niche that can limit access to antibiotics. As current treatment options for M. abscessus infections often fail, there is an urgent need for alternative therapies. Phage therapy is being used to treat M. abscessus infections as an option of last resort. However, little is known about the ability of phages to kill bacteria in the host environment and specifically in an intracellular environment. Here, we demonstrate the ability of phages to enter mammalian cells and to infect and kill intracellular M. abscessus. These findings support the use of phages to treat intracellular bacterial pathogens.

The heterogenous and diverse population of prophages in Mycobacterium genomes.

IMPORTANCE: Mycobacterium species include several human pathogens and mycobacteriophages show potential for therapeutic use to control Mycobacterium infections. However, phage infection profiles vary greatly among Mycobacterium abscessus clinical isolates and phage therapies must be personalized for individual patients. Mycobacterium phage susceptibility is likely determined primarily by accessory parts of bacterial genomes, and we have identified the prophage and phage-related genomic regions across sequenced Mycobacterium strains. The prophages are numerous and diverse, especially in M. abscessus genomes, and provide a potentially rich reservoir of new viruses that can be propagated lytically and used to expand the repertoire of therapeutically useful phages.

PhamClust: a phage genome clustering tool using proteomic equivalence.

IMPORTANCE: Bacteriophage genomes are pervasively mosaic, presenting challenges to describing phage relatedness. Here, we describe PhamClust, a bioinformatic approach for phage genome comparisons that uses a new metric of proteomic equivalence quotient for comparative genomics. PhamClust reliably assorts genomes into groups or clusters of related phages and can subdivide clusters into subclusters. PhamClust is computationally efficient and can readily process thousands of phage genomes. It is also a useful analytic tool for exploring the different types of inter-genome relatedness characteristic of phages in different clusters.

The Cytotoxic Mycobacteriophage Protein Phaedrus gp82 Interacts with and Modulates the Activity of the Host ATPase, MoxR.

Approximately 70% of bacteriophage-encoded proteins are of unknown function. Elucidating these protein functions represents opportunities to discover new phage-host interactions and mechanisms by which the phages modulate host activities. Here, we describe a pipeline for prioritizing phage-encoded proteins for structural analysis and characterize the gp82 protein encoded by mycobacteriophage Phaedrus. Structural and solution studies of gp82 show it is a trimeric protein containing two domains. Co-precipitation studies with the host Mycobacterium smegmatis identified the ATPase MoxR as an interacting partner protein. Phaedrus gp82-MoxR interaction requires the presence of a loop sequence within gp82 that is highly exposed and disordered in the crystallographic structure. We show that Phaedrus gp82 overexpression in M. smegmatis retards the growth of M. smegmatis on solid medium, resulting in a small colony phenotype. Overexpression of gp82 containing a mutant disordered loop or the overexpression of MoxR both rescue this phenotype. Lastly, we show that recombinant gp82 reduces levels of MoxR-mediated ATPase activity in vitro that is required for its chaperone function, and that the disordered loop plays an important role in this phenotype. We conclude that Phaedrus gp82 binds to and reduces mycobacterial MoxR activity, leading to reduced function of host proteins that require MoxR chaperone activity for their normal activity.

Therapeutically useful mycobacteriophages BPs and Muddy require trehalose polyphleates.

Mycobacteriophages show promise as therapeutic agents for non-tuberculous mycobacterium infections. However, little is known about phage recognition of Mycobacterium cell surfaces or mechanisms of phage resistance. We show here that trehalose polyphleates (TPPs)-high-molecular-weight, surface-exposed glycolipids found in some mycobacterial species-are required for infection of Mycobacterium abscessus and Mycobacterium smegmatis by clinically useful phages BPs and Muddy. TPP loss leads to defects in adsorption and infection and confers resistance. Transposon mutagenesis shows that TPP disruption is the primary mechanism for phage resistance. Spontaneous phage resistance occurs through TPP loss by mutation, and some M. abscessus clinical isolates are naturally phage-insensitive due to TPP synthesis gene mutations. Both BPs and Muddy become TPP-independent through single amino acid substitutions in their tail spike proteins, and M. abscessus mutants resistant to TPP-independent phages reveal additional resistance mechanisms. Clinical use of BPs and Muddy TPP-independent mutants should preempt phage resistance caused by TPP loss.

Virion glycosylation influences mycobacteriophage immune recognition.

Glycosylation of eukaryotic virus particles is common and influences their uptake, trafficking, and immune recognition. In contrast, glycosylation of bacteriophage particles has not been reported; phage virions typically do not enter the cytoplasm upon infection, and they do not generally inhabit eukaryotic systems. We show here that several genomically distinct phages of Mycobacteria are modified with glycans attached to the C terminus of capsid and tail tube protein subunits. These O-linked glycans influence antibody production and recognition, shielding viral particles from antibody binding and reducing production of neutralizing antibodies. Glycosylation is mediated by phage-encoded glycosyltransferases, and genomic analysis suggests that they are relatively common among mycobacteriophages. Putative glycosyltransferases are also encoded by some Gordonia and Streptomyces phages, but there is little evidence of glycosylation among the broader phage population. The immune response to glycosylated phage virions in mice suggests that glycosylation may be an advantageous property for phage therapy of Mycobacterium infections.

The problem of Mycobacterium abscessus complex: multi-drug resistance, bacteriophage susceptibility and potential healthcare transmission.

OBJECTIVES: Mycobacterium abscessus complex is responsible for 2.6-13.0% of all non-tuberculous mycobacterial pulmonary infections and these are notoriously difficult to treat due to the complex regimens required, drug resistance and adverse effects. Hence, bacteriophages have been considered in clinical practice as an additional treatment option. Here, we evaluated antibiotic and phage susceptibility profiles of M. abscessus clinical isolates. Whole-genome sequencing (WGS) revealed the phylogenetic relationships, dominant circulating clones (DCCs), the likelihood of patient-to-patient transmission and the presence of prophages. METHODS: Antibiotic susceptibility testing was performed using CLSI breakpoints (n = 95), and plaque assays were used for phage susceptibility testing (subset of n = 88, 35 rough and 53 smooth morphology). WGS was completed using the Illumina platform and analysed using Snippy/snp-dists and Discovery and Extraction of Phages Tool (DEPhT). RESULTS: Amikacin and Tigecycline were the most active drugs (with 2 strains resistant to amikacin, and one strain with Tigecycline MIC of 4 µg/mL). Most strains were resistant to all other drugs tested, with Linezolid and Imipenem showing the least resistance, at 38% (36/95) and 55% (52/95), respectively. Rough colony morphotype strains were more phage-susceptible than smooth strains (77%-27/35 versus 48%-25/53 in the plaque assays, but smooth strains are not killed efficiently by those phages in liquid infection assay). We have also identified 100 resident prophages, some of which were propagated lytically. DCC1 (20%-18/90) and DCC4 (22%-20/90) were observed to be the major clones and WGS identified 6 events of possible patient-to-patient transmission. DISCUSSION: Many strains of M. abscessus complex are intrinsically resistant to available antibiotics and bacteriophages represent an alternative therapeutic option, but only for strains with rough morphology. Further studies are needed to elucidate the role of hospital-borne M. abscessus transmission.

Intrapulmonary Treatment with Mycobacteriophage LysB Rapidly Reduces Mycobacterium abscessus Burden.

Intrinsic and acquired antibiotic resistance in Mycobacterium abscessus presents challenges in infection control, and new therapeutic strategies are needed. Bacteriophage therapy shows promise, but variabilities in M. abscessus phage susceptibility limits its broader utility. We show here that a mycobacteriophage-encoded lysin B (LysB) efficiently and rapidly kills both smooth- and rough-colony morphotype M. abscessus strains and reduces the pulmonary bacterial load in mice. LysB aerosolization presents a plausible treatment for pulmonary M. abscessus infections.

Mycobacterium trehalose polyphleates are required for infection by therapeutically useful mycobacteriophages BPs and Muddy.

Mycobacteriophages are good model systems for understanding their bacterial hosts and show promise as therapeutic agents for nontuberculous mycobacterium infections. However, little is known about phage recognition of Mycobacterium cell surfaces, or mechanisms of phage resistance. We show here that surface-exposed trehalose polyphleates (TPPs) are required for infection of Mycobacterium abscessus and Mycobacterium smegmatis by clinically useful phages BPs and Muddy, and that TPP loss leads to defects in adsorption, infection, and confers resistance. Transposon mutagenesis indicates that TPP loss is the primary mechanism for phage resistance. Spontaneous phage resistance occurs through TPP loss, and some M. abscessus clinical isolates are phage-insensitive due to TPP absence. Both BPs and Muddy become TPP-independent through single amino acid substitutions in their tail spike proteins, and M. abscessus mutants resistant to TPP-independent phages reveal additional resistance mechanisms. Clinical use of BPs and Muddy TPP-independent mutants should preempt phage resistance caused by TPP loss.

Unusual prophages in Mycobacterium abscessus genomes and strain variations in phage susceptibilities.

Mycobacterium abscessus infections are relatively common in patients with cystic fibrosis and are clinically challenging, with frequent intrinsic resistance to antibiotics. Therapeutic treatment with bacteriophages offers some promise but faces many challenges including substantial variation in phage susceptibilities among clinical isolates, and the need to personalize therapies for individual patients. Many strains are not susceptible to any phages or are not efficiently killed by lytic phages, including all smooth colony morphotype strains tested to-date. Here, we analyze a set of new M. abscessus isolates for the genomic relationships, prophage content, spontaneous phage release, and phage susceptibilities. We find that prophages are common in these M. abscessus genomes, but some have unusual arrangements, including tandemly integrated prophages, internal duplications, and they participate in active exchange of polymorphic toxin-immunity cassettes secreted by ESX systems. Relatively few strains are efficiently infected by any mycobacteriophages, and the infection patterns do not reflect the overall phylogenetic relationships of the strains. Characterization of these strains and their phage susceptibility profiles will help to advance the broader application of phage therapies for NTM infections.

Mycobacterial nucleoid-associated protein Lsr2 is required for productive mycobacteriophage infection.

Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.

Phage therapy: From biological mechanisms to future directions.

Increasing antimicrobial resistance rates have revitalized bacteriophage (phage) research, the natural predators of bacteria discovered over 100 years ago. In order to use phages therapeutically, they should (1) preferably be lytic, (2) kill the bacterial host efficiently, and (3) be fully characterized to exclude side effects. Developing therapeutic phages takes a coordinated effort of multiple stakeholders. Herein, we review the state of the art in phage therapy, covering biological mechanisms, clinical applications, remaining challenges, and future directions involving naturally occurring and genetically modified or synthetic phages.

Phage Therapy for Nontuberculous Mycobacteria: Challenges and Opportunities.

Non-tuberculous mycobacterium (NTM) infections are often clinically challenging, with lengthy antibiotic regimens that fail to resolve the infections with few good outcomes remaining. Mycobacteriophages-viruses that infect Mycobacterium hosts-show promise as therapeutic agents for NTM infections and have been used in 20 compassionate use cases. Favorable outcomes were observed in many but not all cases, although the phages show exceptional safety profiles and no evidence of phage resistance was observed, even when only a single phage was administered. Phage-specific antibodies are commonly present following intravenous administration and are often neutralizing for the phage in vitro. However, phage neutralization does not consistently correlate with poor treatment outcomes and may not be a therapeutic limitation in all patients, even when immunocompetent. Currently, the therapeutic potential of phages is substantially limited by the great variation in phage susceptibility and a relatively small repertoire of therapeutically useful phages. As many as 45% of clinical isolates can have a smooth colony morphotype, and phages that both efficiently infect and kill these strains have yet to be described. In contrast, ~ 75% of rough strains are susceptible to and killed by one or more phages and therapeutic options can be considered on a compassionate use basis. Although therapies must currently be personalized, elucidating the determinants of phage host specificity, expanding the useful phage repertoire, and identifying the key determinants of clinical outcomes will reveal their full therapeutic potential.

Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease.

BACKGROUND: Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. METHODS: Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. RESULTS: No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. CONCLUSIONS: Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.

PhaMMseqs: a new pipeline for constructing phage gene phamilies using MMseqs2.

The diversity and mosaic architecture of phage genomes present challenges for whole-genome phylogenies and comparative genomics. There are no universally conserved core genes, ∼70% of phage genes are of unknown function, and phage genomes are replete with small (<500 bp) open reading frames. Assembling sequence-related genes into "phamilies" ("phams") based on amino acid sequence similarity simplifies comparative phage genomics and facilitates representations of phage genome mosaicism. With the rapid and substantial increase in the numbers of sequenced phage genomes, computationally efficient pham assembly is needed, together with strategies for including newly sequenced phage genomes. Here, we describe the Python package PhaMMseqs, which uses MMseqs2 for pham assembly, and we evaluate the key parameters for optimal pham assembly of sequence- and functionally related proteins. PhaMMseqs runs efficiently with only modest hardware requirements and integrates with the pdm_utils package for simple genome entry and export of datasets for evolutionary analyses and phage genome map construction.

Hybrid Vigor: Importance of Hybrid λ Phages in Early Insights in Molecular Biology.

Laboratory-generated hybrids between phage λ and related phages played a seminal role in establishment of the λ model system, which, in turn, served to develop many of the foundational concepts of molecular biology, including gene structure and control. Important λ hybrids with phages 21 and 434 were the earliest of such phages. To understand the biology of these hybrids in full detail, we determined the complete genome sequences of phages 21 and 434. Although both genomes are canonical members of the λ-like phage family, they both carry unsuspected bacterial virulence gene types not previously described in this group of phages. In addition, we determined the sequences of the hybrid phages λ imm21, λ imm434, and λ h434 imm21. These sequences show that the replacements of λ DNA by nonhomologous segments of 21 or 434 DNA occurred through homologous recombination in adjacent sequences that are nearly identical in the parental phages. These five genome sequences correct a number of errors in published sequence fragments of the 21 and 434 genomes, and they point out nine nucleotide differences from Sanger's original λ sequence that are likely present in most extant λ strains in laboratory use today. We discuss the historical importance of these hybrid phages in the development of fundamental tenets of molecular biology and in some of the earliest gene cloning vectors. The 434 and 21 genomes reinforce the conclusion that the genomes of essentially all natural λ-like phages are mosaics of sequence modules from a pool of exchangeable segments.

Nebulized Bacteriophage in a Patient With Refractory Mycobacterium abscessus Lung Disease.

An elderly man with refractory Mycobacterium abscessus lung disease previously developed anti-phage neutralizing antibodies while receiving intravenous phage therapy. Subsequent phage nebulization resulted in transient weight gain, decreased C-reactive protein, and reduced Mycobacterium burden. Weak sputum neutralization may have limited the outcomes, but phage resistance was not a contributing factor.

Mycobacteriophages: From Petri dish to patient.

Mycobacteriophages-bacteriophages infecting Mycobacterium hosts-contribute substantially to our understanding of viral diversity and evolution, provide resources for advancing Mycobacterium genetics, are the basis of high-impact science education programs, and show considerable therapeutic potential. Over 10,000 individual mycobacteriophages have been isolated by high school and undergraduate students using the model organism Mycobacterium smegmatis mc2155 and 2,100 have been completely sequenced, giving a high-resolution view of the phages that infect a single common host strain. The phage genomes are revealed to be highly diverse and architecturally mosaic and are replete with genes of unknown function. Mycobacteriophages have provided many widely used tools for Mycobacterium genetics including integration-proficient vectors and recombineering systems, as well as systems for efficient delivery of reporter genes, transposons, and allelic exchange substrates. The genomic insights and engineering tools have facilitated exploration of phages for treatment of Mycobacterium infections, although their full therapeutic potential has yet to be realized.